Angiogenesis is the development of new blood vessels from existing capillaries. This process has been implicated in a number of human diseases, as well as in the growth and metastasis of solid tumors. In some forms of arthritis, new capillaries form in the joint, leading to its gradual destruction. Solid tumors also must stimulate the formation of new blood vessels in order to obtain the nutrients and oxygen necessary for their growth, thus providing a route by which the tumors can metastasize to distant sites.
Experimental evidence has suggested that malignant tumors can induce angiogenesis through the elaboration of a variety of factors, such as acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor-.alpha. (TNF-.alpha.), and many others (Liotta et al., 1991, Cell 64: 327-336; Hanahan et al., Cell 86: 353-364).
A number of compounds have been discovered or developed which inhibit one or more of the factors or steps involved in angiogenesis. Several of these inhibitors are currently undergoing clinical evaluation in cancer trials. For example, Platelet Factor 4 is a 28 kDa tetrameric protein, which down-regulates aFGF receptors and inhibits endothelial cell proliferation and capillary tube formation. It also inhibits tumor-induced angiogenesis in vivo. The polysaccharide DS4152 inhibits bFGF binding to endothelial cells and cell proliferation, and has been shown to inhibit tumor angiogenesis and growth. FR111142, currently in Phase II cancer trials, is preferentially cytostatic to endothelial cells and inhibits collagenase activity. The identification of additional biologically active agents to inhibit angiogenesis would be highly desirable, because of their potential in providing effective treatment for cancer, arthritis, and other angiogenic diseases.
Members of the Fusarium species are distributed worldwide as soil inhabitants and parasites of cultivated plants. Some isolates of the species are capable of producing mycotoxins, the ingestion of which has been associated with a variety of animal intoxications. For example, chicks fed diets containing a 5% level of crude fungus cultures from Fusarium equiseti develop a syndrome known as Avian Tibial Dyschondroplasia (ATD), characterized by bone deformation and failure of cartilage calcification (Walser et al., 1982, Vet Pathol 19: 544-550).
A water soluble component from Fusarium cultures, fusarochromanone (FC), has been purified and characterized (Pathre et al., 1986, Can. J. Chem 64: 308-311), and one form of this compound, designated FC101 and having the chemical formula: 5-amino-2,2-dimethyl-6-[3'-(R,S) amino-4'-hydroxy-butan-1-one]-2,3-dihydro-4H-1-benzopyran-4-one, has been shown to cause ATD when administered to day-old broiler chicks (Lee et al., 1985, Appl Environ Microbiol 50:102-107).
Various hypotheses have been promulgated to explain the mechanism by which FC101 causes ATD. These include a direct effect on the matrix vesicles, the cell-derived structures which initiate the mineral formation necessary for growth plate calcification, or an inhibitory effect on the ability of growth plate chondrocytes to deposit mineral.
The vascular invasion of growth plate cartilage, also known as neovascularization or angiogenesis, is a process of new capillary formation from pre-existing vessels. The process involves several steps, including the degradation of the extracellular matrix around a local venule; migration of the endothelial cells (EC) lining the inner wall of blood vessels; proliferation of the EC; the resulting formation of capillary tubes; and, finally, the survival and maturation of newly formed blood vessels (Benjamin and Keshet, 1997, Proc. Natl. Acad. Sci. USA 94:8761-8766; Brooks et al., 1994, Cell 79:1157-1164). The present inventors hypothesized that the underlying pathogenicity of ATD results from a defect in the vascularization in affected areas of growth plate cartilage, although the reasons for this defective vascularization were unknown (Nie et al.,1995, J Bone Miner Res 10: 1625-1634).
The present invention relates to the discovery that a water soluble compound extracted from fungal cultures demonstrates a selective toxicity towards endothelial cells. This selective toxicity inhibits angiogenesis, and thus prevents capillary formation from pre-existing blood vessels.
In addition to fusarochromanone production, some members of the Fusarium genus have previously been shown to produce a number of biologically active compounds; some useful as pharmaceutical agents. Sawada et al, in U.S. Pat. No. 4,579,819, described a method for the production of ursodeoxycholic acid, useful in the therapy of cholesterol gallstones, by using microbial transformation with members of the Fusarium genus. Burmeister, in U.S. Pat. No. 3,959,468, disclosed an antibiotic produced by Fusarium equiseti, which was shown to have biological activity against several genera of gram negative bacteria.
Biological agents have also been extracted from the mycelium of a Fusarium. Specifically, Simon-Lavoine et al, in U.S. Pat. No. 4,233,291, disclosed a depsipeptide extracted from Fusarium equiseti, which stimulated the body's defenses when administered in pharmaceutical compositions. The depsipeptide was particularly useful in human and veterinary therapy for the treatment of chronic or acute respiratory diseases, such as chronic bronchitis and emphysema. Additionally, intraperitoneal administration of 50 .mu.g of the depsipeptide was shown to increase the survival time of mice previously inoculated with 1000 L1210 leukemic cells and treated with cyclophosphamide.
Anti-leukemic activity and low toxicity in mice was also demonstrated for a novel antibiotic disclosed in Japanese Application No. 4036-276-A from Tayca Corporation. However, neither the molecular structure of this compound, nor that of the depsipeptide disclosed by Simon-Lavoine et al, is identical to that of FC101.